Method of carrying out enzyme-catalyzed reactions

ABSTRACT

A liquid stream containing the reactants is passed through a bed of enzyme catalyst, made up of enzymes bonded to small, dense particles of carrier or support materials, in a manner which causes the bed to expand or fluidize and a chemical reaction is thereby carried out in a process that is simultaneously free from limitations due to plugging and excessive pressure drop and which also has the advantage of the high mass transfer rates that can be realized between the liquid and small particles.

United States Patent [191 Coughlin et al.

[ Dec. 23, 1975 METHOD OF CARRYING OUT ENZYME-CATALYZED REACTIONS [76] Inventors: Robert W. Coughlin, 902 Seventh Ave., Bethlehem, Pa. 18018 [22] Filed: Feb. 23, 1972 [21] Appl. No.: 228,748

OTHER PUBLICATIONS Emery et al., Some Applications of Solid-Phase Enzymes in Biological Engineering, Birmingham Univ.

Chemical Engineer, Vol. 22, No. 2, (Summer 1971), pp. 37-41.

MPEP 201.03, Rules of Practice, pp. 23,24.

Barker et al., Process Biochemistry, Vol. 6, No. 10, (1971), pp. 11-13.

Leva, Fluidization, pp. 6-11, 291-296, McGraw Hill, (1959).

Primary ExaminerA. Louis Monacell Assistant Examiner-Thomas G. Wiseman' [57] ABSTRACT A liquid stream containing the reactants is passed through a bed of enzyme catalyst, made up of enzymes bonded to small, dense particles of carrier or support materials, in a manner which causes the bed to expand or fluidize and a chemical reaction is thereby carried out in a process that is simultaneously free from limitations due to plugging and excessive pressure drop and which also has the advantage of the high mass transfer rates that can be realized between the liquid and small particles.

2 Claims, N0 Drawings METHOD OF CARRYING OUT ENZYME-CATALYZED REACTIONS BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a method for carrying out chemical reactions using as catalysts enzymes that are insolubilized or immobilized by bonding them to solid supporting materials, and in which a solution of the chemical reactants passes upward through a bed of such solid supporting material to which the enzymes of choice have been bound, such supporting material being in the form of particles of the proper size and density and the reactant solution flowing through the bed at such a rate that the bed of particles becomes expanded or fluidized and the advantages of fluidized bed operations are conferred upon the system and process thereby realized.

2. Description of Prior Art Within the last decade the art of attaching enzymes to insoluble supporting materials has been developed and such solid-bound enzyme catalysts can now be applied to practical, commercial processes such as producing glucose from starch, hydrolyzing proteins and sugars, clarifying fruit juices and beer, carrying out reactions for producing antibiotic pharmaceutical. reagents, treating human blood for promoting desired chemical reactions such as decomposition of urea, and many other useful and potentially useful purposes. A major advantage of using enzymes in such insoluble form bound to a solid supporting material is that the catalytically active enzyme may be physically retained in the reaction vessel and contacted there with a continuously flowing liquid process stream. Before it was possible to bond enzymes to such insoluble supports, the enzyme would remain in the liquid process stream or could be separated therefrom only with difficulty with the result that the enzyme could be reused only with difficulty or not at all. Now that the art exists for bonding enzymes to insoluble supports, it becomes possible to use them in much the same way as ordinary heterogeneous catalysts on inert, insoluble supporting carriers that are well known in the chemical process industry. Such use now permits the continuous, convenient reuse of the same insolubilized enzyme catalyst for contacting a continuously flowing liquid reaction process stream thereby catalyzing a desired chemical reaction within said stream but without the necessity of separating the enzyme from the reaction products or the possible disadvantage of losing said enzyme entirely and not being able to re-use it.

However, some major problems have been encountered in the practical application of such insolubilized enzymes. They have usually been bonded to natural and synthetic high polymers and then used in fixed bed reactors; in such systems disadvantageously high pressure drop and plugging have been encountered and this behavior can be attributed to the small particle size and to the deformable, gel-like properties of the polymeric supporting material. More recent attempts to circumvent these kinds of problems have involved using inorganic supporting materials such as apatite and glass. While the latter materials are less deformable than the organic polymeric supports, columns packed with these materials still are susceptible to plugging and cause high pressure drop when small particle sizes are used in fixed, packed beds. When the particles are made sufficiently large to avoid such adverse plugging and pressure-drop behavior, their very size causes large mass transfer resistances both within the liquid film surrounding the support particles and within the support particles themselves if they are porous. Such mass transfer resistance can prevent the reactants from reaching the immobilized enzyme as fast as they can react and thereby can result in inefficient utilization of the enzyme bound to the solid support. To minimize mass transfer resistances, both within the solid supporting particles themselves and in the liquid film surrounding them, the particles should be as small as possible. However, as has already been stated, fixed packed beds of small particles are markedly susceptible to plugging and cause disadvantageously high pressure drops. Stirred-tank, slurry reactors are also susceptible to plugging and have the added disadvantages of back mixing, mechanical complexity and high shear rates.

Until the present invention there has been no simple way of utilizing insolubilized enzymes bound to solid supporting materials in a process that simultaneously provides relative freedom from plugging and from high pressure drops, reasonably high liquid flow rates in approximate plug flow and excellent mass transfer rates from process stream to the solid supporting particles and within the particles themselves. The present invention employs an expanded orfluidized bed of insoluble support particles to which enzyme is bound but the present invention differs from prior art in that expanded or fluidized bed processing has never before been applied to enzyme catalyzed reactions, in that the insoluble, support particles must have certain types of properties for good fluidization behavior in a fast flowing liquid stream, and in that said good fluidization properties are achieved by constructing the insolubilized-enzyme, catalyst particles in new and different ways.

SUMMARY OF THE INVENTION It is an object of this invention to provide a means of carrying out enzyme-catalyzed reactions by a process which is not susceptible to plugging, which permits large flow rates of liquid reactant stream with low pressure drop and which employs enzymes bound to solid supporting material as carrier.

It is another object of this invention to provide a process with the above-described advantages and for the purpose stated above, that can also operate using small particles of supporting material to which enzyme catalyst is bound and which thereby provides high rates of mass transfer from the liquid stream to the enzyme catalyst particles and within these'particles.

It is a further object of this invention to provide such a process that also permits approximate plug. flow with little backmixing of the liquid as it flows through the bed of enzymic catalyst particles.

Still another object of this invention is to provide insolubilized enzyme catalysts having properties of density and particle size suitable for conducting such processes as described above.

Yet another object of this invention is to provide methods of preparing insolubilized enzyme catalysts possessing such properties.

These and other objects have now herein been attained by a process in which the enzymes are insolubilized by binding them to the outer surfaces of non-porous, dense, inorganic particles, or to a thin layer or organic polymer or of glass on the outside surfaces of such particles, passing a steady stream of liquid reactant through a bed of such particles, the flow rate, viscosity and density of the liquid and the sizes and densities of the particles being of the values required to cause expansion or fluidization of the bed of particles and thereby providing the benefits of fluidized or expanded bed operation with a liquid stream flowing in approximate plug flow through the fluidized or expanded bed.

DEFINITIONS Within this disclosure, the term expanded bed refers to a process wherein the particles of the bed are suspended and agitated by the liquid but do not mix or circulate within the bed to any appreciable extent, the term fluidized bed to a process wherein the suspended and agitated particles do circulate and mix within the bed, and the term suspended bed is generic to the terms fluidized bed and expanded bed and used to refer to either one or both of them.

The term enzyme as used within this disclosure can refer to an enzyme, a coenzyme or an enzyme-analogue (which is a molecule synthesized to approximate the catalytically active structure of a natural enzyme and which has similar catalytic activity). The terms immobilized and insolubilized are used interchangeably throughout this disclosure to denote either chemical or physical binding of an enzyme to a carrier or entrapment of an enzyme within a carrier.

DESCRIPTION OF PREFERRED EMBODIMENTS The embodiments discussed here are presented herein for purposes of illustration only and are not intended to be limiting in any manner.

According to the present invention, one of the important properties of a useful insolubilized enzyme catalyst support or carrier is a density sufficiently large to permit the use of small particles and large liquid flow rates in expanded-or fluidized-bed operation. Although insolubilized enzymes have been known or used for more than a decade, they have never before been employed in expanded or fluidized bed processes; one reason for this undoubtedly is the fact that in almost every instance the enzymes have been bound to materials of low density. The present invention calls for the use of particles of inert, high density materials such as noncorrodible metals or metal oxides as supporting material to which the enzyme or enzymes of choice are then bound.

The carrier or support for the enzymes as used in this invention can be particles of metallic nickel or nickel oxide sinter as supplied by the International Nickel Company, Inc. under the trade names Nickel Oxide Sinter 75 and Nickel Oxide Sinter 90. The density of this material is about 8 gm/cm and an appropriate particle-size size range is 10m to 0.25 inch for fluidization by water, although the preferred size range will depend on the density and viscosity of the fluidizing liquid. Enzymes may be bound to the nickel oxide material directly; metallic nickel particles can be partially oxidized in order to develop a coating of nickel oxide on their surface by exposing them to an air or oxygen atmosphere at high temperature. As reported by Weetall and coworkers [Biochim. Biophys Acta 206, pp 54-60 (1970)] an alkylaminosilane derivative of the oxide may then be prepared by refluxing the oxide coated support particles about 24 hours in a toluene solution containing about 10 percent 'y-aminopropyltriethoxysilane. The particles are then washed in solvents such as toluene and acetone, dried under vacuum at 60C, and then the alkylamino group on the particles is acylated by refluxing for about 24 hours in a chloroform solution containing 10 percent p-nitrobenzoyl chloride and 10 percent triethyl amine. After washing with chloroform and drying at 60C under vacuum the aryl nitro group and the particles is reduced to an aryl amino group by refluxing for about 1 hour in an aqueous solution of 10 percent sodium dithionite. After washing with water and drying, the particles are then diazotized by reaction with an aqueous solution containing about 2.5 percent of sodium nitrite in 2N I-ICl under conditions of 0C and gentle aspiration to remove evolved NO This reaction is complete after about 20 minutes whereupon the particles are filtered and washed with cold aqueous sulfamic acid (about 1 percent) and then immediately contacted with an appropriate buffer solution of the enzyme of choice which is thereby coupled to the particle by a diazo type bond. This diazo type bond hyad been used successfully to insolubilize L-amino acid oxidase [Weetall and Baum, Biotech. & Bioeng. XII, pp. 399-407 (1970)], urease [Weetall and Hersh, Biochim. Biophys Acta 185, pp. 464-465 (1969)], alkaline phosphatase ]Weetall, Nature 223, p. 959 (1969)], trypsin and papain [Weetall, Science 166, pp. 615-617 (1969)]and nicotinamide adenine dinucleotide (NAD) [Weibe], et al, Biochem and Biophys Res Comm 44, pp 347-352 (1971)]. In each of these cases and enzyme coupling reaction should be carried out under mild conditions: aqueous medium, low temperature and a pH near the neutral point. After coupling to the enzyme the particles are used in a liquid fluidized bed reactor.

Another type of chemical bond that can be used to couple an enzyme to a metal oxide is that formed between the enzyme and an isothiocyanate group attached to the solid. To do this the metal oxide is refluxed as described above with 'y-aminopropyltriethoxysilane solution in toluene and the resulting alkylamine derivative is then refluxed overnight in a 10 percent solution of thiophosgene in chloroform, washed with chloroform, dried and immediately contacted with an appropriately buffered solution of the enzyme of interest. This type of chemical bonding has been used to immobilize trypsin, ficin, papain and glucose oxidase [Weetall, Biochem. Biophys. Acta 212, pp. 1-7 (1970)] and several other enzymes [Manecke, Biochem. Journal 107, pp. 2-3P, (1968)]. Various techniques for bonding a variety of enzymes to inorganic carriers using silane coupling agents have been disclosed and claimed in US. Pat. No. 3,519,538 granted to Messing et al.

It is evident that many other kinds of chemical bonds can be employed to attach enzymes to a dense, particulate carrier suitable for expanded-or fluidized-bed operation without departing from the substance of the present invention; the various types of chemical bond that have been successfully employed are part of the established art and are described in published literature [e.g. Kay, Process Biochemistry 3, 36 (1968) and Brown et a1, Enzymologia 35, 215 (1968)]. It is also evident that it is possible to practice the present invention using various other types of dense particulate material to which the enzyme of choice is then bound. By proper selection of these materials it is possible to vary the density of the insolubilized enzyme catalyst particles over a wide range; for example it is possible to use glass (density 2.5 gm/cm), nickel (density 8.9 gm/cm iron or preferably stainless steel (density about 8 gm/cm silver (density 10.5 gmlcm molybdenum (density 10.2 gmlcm tantalum (density 16.6 gm/cm gold or tungsten (density 19.3 gm/cm rhodium (density 21.4 gm/cm platinum (density 21.5 gin/cm) and various alloys of these metals. Various oxides, hydroxides, carbides, silicates and other insoluble chemical compounds of dense chemical elements may also be used as such carriers. Clearly, the metals, alloys or similar materials should also be chosen for inertness, resistance to the particular aqueous environment to be employed and for properties of non-interference with the particular enzyme and chemical reaction system to be employed. It is evident that a wide variety of materials can be used for these carrier particles.

When it is possible to form a tough, tenacious, insoluble and otherwise suitable oxide coating on metallic particles of the proper density, the enzyme of choice can be bound to the oxide by one of the techniques outlined above in which the oxide is silanized to produce alkylsilane groups on the surface as a first step. it is clear that it is also possible to use particles of the metal oxide itself as the carrier material using these procedures. Another possible approach, however, is to first coat the particles with a suitable polymer and then use one of the many known techniques for chemically bonding the enzyme of choice to the polymer. These techniques for bonding enzymes to polymers, which are part of the established art, have been discussed by Goldstein and Katchalski [Zeitschrift fur analytische Chemie 243, p. 375 (1968)] and Manecke [Proceedings of the Biochemical Society, page 2 P, (January 1968)] and they include the use of derivatives of polymers such as cellulose, polystyrene, copolymers of leucine and phenylalanine, copolymers of methacrylic acid, methylacryl-3-fluoroanilide and divinyl benzene, copolymers of methacrylic acid, fluorostyrene and divinyl benzene, polymers of m-isothiocyanatostyrene and vinylisothiocyanate and chemical bonds such as azide, diazo, isocyanato, isothiocyanato, carbodiimide and sulfonamide; enzymes that have been bound by these techniques include glucose oxidase, papain, ficin, trypsin, urease, diastase, invertase, chymotrypsin and alcohol dehydrogenase. An alternative but similar approach is to coat the dense carrier particles with glass by using the fine particle glazes and sealants supplied by Owens-Illinois and by Corning Glass and then to bond the enzyme of choice to this glass layer using one of the techniques described above for bonding enzymes to metallic oxides that begins by refluxing the glasscoated particles with a toluene solution of 'y-aminopropyltriethoxysilane.

There are many procedures known in the art to coat dense particles (such as of a metal or a metallic oxide with layers of polymer or of glass. Almost any recognized film formingtechnique can be used for coating with a polymer. For example, the particles can be simply contacted with a solution of the polymer of choice, after which the particles are filtered and dried to remove the solvent and leave a layer of polymer on their surfaces. An alternative approach is to produce the polymer or copolymer by polymerization within a solution in which the particles of choice are suspended; as the polymer forms and grows in solution it will tend to deposit on the particles which can then be filtered and dried. In this latter technique it is possible to control the molecular weight and porosity of the polymer deposited on the particles by controlling temperature and concentrations of monomer, solvent and crosslinking agent present in the mixture. Adhesion between the dense particles and the polymer can often be improved by first coating with a bonding latex such as the Accobond resins supplied by the American Cyanamid Company, in accord with the manufacturers instructions. To coat metallic particles with glass they cdn be contacted with a suspension of a glass frit such as the sealants mentioned above which have particles which pass a 325 mesh screen; then the metal particles coated with the glass particles are dried and baked in a furnace to bond the glass to the metal; it is possible to control the porosity and particle size of the resulting layer of glass on the metal by proper choice of glass frit size and baking temperature.

In addition to chemical attachment of enzymes to polymers, glass and solid oxides it is also possible to prepare water-insoluble derivatives of enzymes by physical adsorption onto colloidal particles, by entrapment of enzymes within insoluble matrices of crosslinked polymers and by chemical crosslinking of an enzyme by a bifunctional reagent. All of these techniques for preparing water-insoluble derivatives of enzymes are known in the art and have been described in the published chemical literature. It will be obvious to one possessing ordinary skill in the art how to adapt these various techniques for enzyme insolubilization to forming thin films of enzyme or enzyme-bearing material on the surfaces of small, dense particles suitable for use in the present invention. For example, colloidal particles may first be bonded to the surface of the dense particles suitable for fluidization in the present invention followed by adsorption of enzyme within the porous layer of colloidal particles thus formed. Alternatively, cross-linked polymers containing entrapped enzymes may be bonded to the dense, small particles specified in the present invention or these particles may be coated with an insoluble layer of cross-linked enzymes.

Preparation .of enzyme catalysts immobilized by bonding to dense particles as described herein permits their use in expanded-bed or fluidized-bed reactors in which the particles are maintained in fluidized suspension by the flow of a stream of liquid (which contains the substrate or reactant) through the bed. Such liquid fluidization provides the advantages of approximate plug flow of the liquid through the bed in contrast to a stirred, slurry-type reactor and also relative freedom from plugging and from high pressure drop in contrast to fixed-bed operation. In the usual type of fluidized operation the fluidized particles will be well mixed within the bed but it is possible to avoid the mixing of the particles from one part of the bed to another by packing the bed with larger particles which do not move or mix themselves and thereby prevent the small enzyme-bearing particles from mixing within the bed, but at the same time permit the fluidization of the small particles within the interstices of the packed large particles. Such packed, fluidized beds as described by Gabor [Chem. Eng. Progr. Symp. Series, No.62, 62, 302 (1966)] and Gabor et al. [Chem. Eng. Progr. Symp. Series No. 42, 60, 96 1964)] also fall within the purview of the present invention. I

The use of dense materials for the particles which carry the enzyme permits good fluidization while using particles of small size; using such small particles increases mass-transfer rates of reactants to the particles and therefore also increases the rate of chemical reaction. Furthermore, the use of dense, enzyme-carrier particles has the added advantage of permitting high liquid flow rates through the expandedor fluidizedbed without entrainment and loss of the particles from the bed. The higher liquid flow rates and higher rates of reaction that can be realized in this kind of system using small, dense particles as catalyst carrier provides still another important advantage of high production rate of reaction product from this type of reactor. These and other advantages will be more apparent from the examples which follow.

Having generally described the invention, a further understanding can be obtained by reference to certain specific examples which are presented herein for purposes of illustration only and are not to be limiting in any manner.

8 flow rate of said liquid reactant solution is 70,000 lb/hr, ft of reactor cross section and that the said liquid reactant solution has viscosity equal to 1 centipoise and density equal to 1 gm/cm", the following results are computed for different particle sizes, different particle densities, and two different types of reactor:

1. A fixed-bed reactor in which the particles of enzymic catalyst remain packed in an immobile bed, and

2. fluidized-bed reactors in which the particles of enzymic catalyst are suspended and agitated by the motion of the liquid through the reactor.

EXAMPLE I Conversion of ethanol by reactor 99.9 percent, Schmidt Number 1000, Viscosity of reactant feed l centipoise, Density of reactant feed 1 gm/cm, Mass flow rate of reactant feed 70,000 lb/hr, ft of reactor cross section.

EXAMPLES The superiority of suspended or fluidized-bed operation of liquid plug-flow reactors using as catalysts enzymes bound to small, dense particles is demonstrated herein by design calculations for a reaction, the global or net rate of which is about equal to the rate of mass transfer of the reactant from the bulk solution to the surface of the solid, enzyme-bearing particles; i.e. for a situation in" which the rate of chemical reaction is controlled by mass transfer to the catalytic particle. These computations have been carried out for reaction of a dilute aqueous solution of ethanol containing excess NAD using the Ergun Equation for pressure drop in flow through beds of solid particles [see Principles of Unit Operations by Foust et al, p.475, equation 22.86, John Wiley & Sons, Inc. (1960) or Unit Operations of Chemical Engineering by McCabe and Smith (2nd ed.) p.161, equation 7-26, McGraw-Hill Book Company (1967)], correlations for mass transfer coefficients in fixed and in packed beds of particles [see Chemical Engineering Kinetics by J. M. Smith, pp. 380-383, equations 10-36 through 10-39 and FIG. 10-2 on p.364, McGraw-Hill Book Company (1970)], a smoothed correlation of particulate fluidization [see Fluidization and Fluid-Particle Systems by Zener and Othmer, p. 236, Figure 7.7, Reihold Publishing Co. (1960)], and the common, well known design equation for plug-flow reactors. Assuming that the reactant is ethanol in aqueous solution in which the diffusivity of ethanol is about 10- cm /sec [i.e. a dimensionless Schmidt Number equal to 1000 as would be the case for a dilute aqueous solution of ethanol reactant], that the reactor effects a conversion of said ethanol reactant equal to 99.9 percent, that the liquid reactant solution is passed in plug flow through beds of approximately spherical particles of approximately uniform diameter in each type of reactor, and assuming further that the It is to be noted for the results of Example I that the reactor length necessary to carry out the specified reaction is an order of magnitude larger when a fixed bed reactor packed with catalyst particles of about 5300 .Lm or 0.53 cm diameter is employed in contrast to the reactor lengths required for fluidized-bed reactors containing catalyst particles of about 500;rm or 300p.m diameter. With regard to these same results it is also noteworthy that the pressure drop required for the fluidized-bed reactor for this reaction is between 6- and l2-times lower than for the fixed-bed reactor.

It has been demonstrated, therefore, that, compared to a fixed bed reactor carrying out the identical reaction, and fluidized bed reactors possess the dual advantages of much lower pressure drop and much smaller size. The required length and pressure drop of the fixed-bed reactor could be lowered somewhat by packing it with smaller particles of enzymic catalyst but this would make the reactor far more susceptible to plugging which would eventually lead to still larger pressure drops across this reactor. Likewise, the pressure drop across the fixed bed reactor could be diminished somewhat by packing it with larger catalytic particles but this would increase still further the length of the reactor required for the stated conversion of reactant. With regard to particle size and density, for the feed flow rate, density and viscosity of the present example, it would be impossible to use 500,um diameter particles of density 2.5 gm/cm or lower, or even 300um diameter particles of density 8.6 gm/cm or lower in fluidized-bed operation; of this were attempted the catalyst particle's would be swept from the reactor; however, denser particles can be kept in the reactors under these conditions. This clearly demonstrates the advantages of using catalysts comprising enzymes attached to very dense particles to permit the use of small particles in these kinds of fluidized reactors. The advantages of the flui- EXAMPLE II Reactor length 1 foot; Schmidt Number 1000 Viscosity of reactant feed l centipoise, Density of reactant feed 1 gm/cm, Mass flow rate of reactant feed 70,000; lb/hr, ft of reactor cross section.

ber to make it conform with that reactant which limits the 'rate of reaction by its rate of transport from the bulk solution to the surface of the solid.

In practice a suitable representative configuration for the fluidized-bed reactor in the instant Examples is a glass or metal tube in a vertical position with a screen or support grid spanning the lumen of the tube near the bottom. The openings of the supporting screen or grid should be sufficiently small to prevent passage of the particles of the bed but not so small as to cause unnecessarily large pressure drops. Appropriate sizes for the instant Examples are holes of about 250p.m in the support grid and a reactor-tube diameter of about 4 inches.

The particles of enzyme catalyst are placed in the tube and are supported by the grid when the reactor is not in Example ll demonstrates that much larger conversion of reactant can be accomplished by fluidized-bed reactors using enzymic catalyst bound to dense particles of diameters 300p.m and 500 pun, in contrast to a fixed-bed reactor packed with catalytic particles of about 5300;.tm diameter. If particle size were lowered or length increased for the fixed-bed reactor of Example II in order to improve the conversion, the pressure drop would quickly become disadvantageously large. Example ll further demonstrates the increases in conversion that can be obtained in a fluidized reactor by decreasing the size and increasing the density of the particles to which the enzymic catalyst is bound. As in the case if Example I, the advantages of fluidized-bed operation and of dense particles of insolubilized enzymic catalyst would be further amplified for Example II also if more viscous reactant feed were employed.

In the examples described herein the reactant or substrate has been specified as ethanol and the computed results have been based on a rate of chemical reaction assumed equal to the rate at which ethanol is transported from the bulk solution to the surfaces of the enzyme-bearing particles where chemical reaction takes place. A suitable reaction would be the dehydrogenation of ethanol for which an alcohol dehydrogenase enzyme could be bound to the partilces. As mentioned elsewhere in this specification alcohol dehydrogenase has been successfully bound to various polymeric carrier materials so a suitable enzyme carrier could be formed by a metal particle coated with an appropriate polymer. Clearly, if another reactant must react with the substrate (e. g. NAD with ethanol in the instant Examples) it, too, must be present in sufficient concentration in the solution. Other substrates and reactions can be substituted for the alcohol dehydrogenation of the instant Examples, provided the appropriate enzymes are also substituted as the catalysts and the necessary reactants or coenzymes are also present in the feed solution in sufficient concentration. To compute the results for another such reaction system it is necessary to modify the diffusivity or Schmidt Numoperation. Reactions are carried out under fluidization conditions by passing the feed solution upwards through the reactor at the appropriate flow rate.

The fluidizedor expanded-bed reactors in which enzyme-bearing catalytic particles are maintained in a state of agitation and suspension, and in many cases caused to mix or circulate within the reaction zone, by the flow of liquid, as set forth in the present invention, can be used for a variety of chemical processing applications. For example, the reactor systems of the present invention can be used for converting starch to glucose, for inverting sugars, for oxidizing glucose, for decomposing urea or hydrogen peroxide, for lysing lipids, proteins, peptides and other molecules, for dehydrogenating alcohol, for oxidizing lipids, and for many other uses. They should find application for carrying out any enzyme-catalyzed reaction for which the enzyme can be immobilized on a solid, particulate carrier material.

Having fully described the invention, it will be apparent to one having ordinary skill in the art that many modifications and changes can be made without departing from the spirit or scope thereof. For example it is possible to carry out the present invention for many different types of enzyme-catalyzed reactions, using reactors of many different types of construction and mechanical design and by preparing many-different kinds of enzyme-bearing particles of appropriate small particle size and high density suitable for use in such reactors. It is also possible to carry out the present invention while simultaneously removing particles of insolubilized-enzyme catalyst from the reactor, regenerating said catalyst particles in a separate vessel and returning said regenerated catalyst particles to the reactor.

What is claimed and intended to be covered by letters Patent is:

l. A method of carrying out enzymatic reactions using enzymes attached to insoluble, finely divided particles of carrier material which comprises continuously flowing a liquid containing the substrate or reac- 11 12 tant upwardly through a reaction zone containing said said reaction zone. enzyme-bearing particles and maintaining the vertical 2. The method according to claim 1 wherein said liquid velocity within said zone sufficiently high to particles also mix and circulate within said reaction cause suspension and agitation of said particles but zone.

sufficiently low to allow said particles to remain within 

1. A METHOD OF CARRYING OUT ENZYMATIC REACTIONS USING ENZYMES ATTACHED TO INSOLUBLE, FINELY DIVIDED PARTICLES OF CARRIER MATERIAL WHICH COMPRISES CONTINUOUSLY FLOWING A LIQUID CONTAINING THE SUBSTRATE OR REACTANT UPWARDLY THROUGH A REACTION ZONE CONTAINING SAID ENZYME-BEARING PARTICLES AND MAINTAINING THE VERTICAL LIQUID VELOCITY WITHIN SAID ZONE SUFFICIENTLY HIGH TO CAUSE SUSPENSION AND AGITATION OF SAID PARTICLES BUT SUFFICIENTLY LOW TO ALLOW SAID PARTICLES TO REMAIN WITHIN SAID REACTION ZONE.
 2. The method according to claim 1 wherein said particles also mix and circulate within said reaction zone. 